Method for producing dry extracts

ABSTRACT

The invention relates to a method for producing dry extracts of plants and to pharmaceutical preparations containing the same, more particularly phytopharmaceuticals, which contain at least one ethanolic/aqueous extract of a plant (drug), the plants being selected from the group consisting of:  Rumicis herba; Verbena officinalis; Sambucus nigra; Primula veris ; and  Gentiana lutea  and mixtures thereof. The invention further relates to a pharmaceutical for treating inflammatory and/or infectious diseases of the nose and throat area and/or the nasal sinuses, as well as the use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. §371)of PCT/EP2012/066212, filed Aug. 20, 2012, which claims benefit ofEuropean application 11178206.6, filed Aug. 19, 2011, Europeanapplication 11193734.8, filed Dec. 15, 2011, and European application12170125.4, filed May 30, 2012.

FIELD OF THE INVENTION

The invention relates to a method for producing dry plant extracts andto pharmaceutical preparations containing the same, in particularphytopharmaceuticals, which contain at least one ethanolic/aqueousextract of a plant (drug), wherein the plants are selected from thegroup consisting of: Rumex acetosa L., Rumex acetosella L., Rumexobtusifolius L., Rumex patientia L., and Rumex crispus L., (referred tohereinafter by the collective term “Rumicis herba”); Verbenaofficinalis; Sambucus nigra; Primula veris; and Gentiana lutea andmixtures thereof. The invention also relates to a pharmaceutical fortreating inflammatory and/or infectious diseases of the nose and throatarea and/or of the nasal sinuses, and to a dietary supplement, and alsoto the use thereof.

BACKGROUND OF THE INVENTION

The above medicinal plants are known as secretolytic agents in the caseof infections of the upper airways, in particular in the case ofsinusitis. Here, each individual drug contributes its share to theunique efficacy of the composition:

The root of Gentiana lutea (Gentian root) is generally used formedicinal purposes. Various secoiridoid glycosides with expectoranteffect are found inter alia among the ingredients.

The leaves and stem of the aforementioned Rumex types, referred tohereinafter as “Rumicis herba” (Sorrel herb), are generally used formedicinal purposes. Flavonoids and various tanning agents are foundherein as ingredients with anti-inflammatory effect, which additionallypositively support the body's own defences.

The collective term “Rumicis herba (Sorrel herb)” is to be understood tomean a mixture of the following species:

Rumex acetosa L., synonym: Lapathum acetosa SCOP, synonym: Lapathumpratense LAM, synonym: Acetosa pratensis MILLER; Rumex acetosella L,synonym: Rumex infestus SALISB; Rumex obtusifolius L., synonym Lapathumobtusifolium MOENCH, synonym Lapathum obtusantum MONTAD, synonym Rumexactus WALLR, synonym Rumex silvestris WALLR;Rumex patientia L., synonym Rumex olympicus BOISS., synonym Lapathumhortense MOENCH; Rumex crispus L.;Rumex thyrsiflorus FINGERH., synonym Acetosa thyrsiflora FINGERH,synonym Rumex acetosa subsp. auriculatus WALLR.

The leaves and stem of Verbena officinalis (Verbena herb) are preferablyused for medicinal purposes and contain iridoid glycosides,phenylethanoid glycosides and flavonoids as primary ingredients, wherebyexpectorant and antiviral effects are achieved.

The leaves of Sambucus nigra (Elder flower, Sambucus nigra leaves) aretypically used for medicinal purposes, of which the contents containvarious flavonol glycosides and which contains sambunigrin, a cyanogenicglycoside, as a primary ingredient, which have an expectorant andantiviral effect (Grabovac, A. and Ullmer, A., ÖsterreichischeApotheker-Verlagsgesellschaft m.b.H (Austrian Pharmacists' PublishingHouse), 2003).

Flowers and sepals of Primula veris (Primula flower, Primula verisleaves) are used for medicinal purposes. The ingredients comprisetriterpene saponins and also phenol glycosides such as primulayerin.They have an expectorant effect and fight against viruses. Theingredients act as a mild secretolytic agent and expectorant in the caseof treatment of respiratory diseases.

The combination of the aforementioned medicinal plants is known as asecretolytic agent under the trade name Sinupret®, which is registeredfor the applicant and has been available on the market for approximately75 years. The medicinal plants used in Sinupret® are selected, testedand further processed in a targeted manner. The uniform quality of thepharmaceutical attained hereby is achieved by the manufacturer, thecompany BIONORICA, by optimized propagation and harvesting strategiesand also by strict quality control.

The composition forming the basis of Sinupret® is preferably effectivein the case of inflammation and also infections of the throat, nose andear region, and is particularly suitable for the treatment of acute andchronic sinusitis and/or rhinosinusitis.

Both acute and chronic sinusitis are common. In three of four cases, thesinusitis develops as a result of a cold that spreads to the nasalsinuses and is accompanied by inflammation of the mucous membrane. Theairways reach from the main nasal cavity to the various sinuses as faras the pulmonary alveoli. The nasal sinuses include the frontal sinus,the ethmoid sinus, the sphenoid sinus and the maxillary sinus. All ofthe aforementioned bone cavities are lined with mucous membrane and openout via narrow openings (the ostia) into the main nasal cavity.

The surface of the airways is coated with a protective mucus, to whichdirt particles and pathogens, such as viruses, bacteria or funguses,which infiltrate together with the inhaled air, remain adhered. Themucus contains antibodies, which attack the infiltrating substances andmake them harmless. So that the foreign substances can be flushed outfrom the body, the mucus is generally transported away with the aid ofthe cilia of the ciliated epithelium in the direction of the throat,where it can be swallowed. In order to fend off infection-inducedrespiratory diseases, the mucous membrane must have unhinderedprotective and cleaning mechanisms. In order to transport away the mucuscharged with pathogens, the unhindered function of the cilia isindispensible, said cilia transporting the mucus further as a result ofundulating movements. With infection and with inflammatory processes ofthe upper airways, the function of the protective and cleaningmechanisms of the mucous membrane is limited.

For example, viruses such as rhinoviruses, adenoviruses or coronavirusestrigger inflammatory reactions of the mucous membranes, whereby themucous membrane becomes swollen and produces increased mucus. Thisinitially results in aqueous and then viscous mucus flow. Over thecourse of inflammation of the nasal mucous membrane, the ostia of thesinuses may swell and impair or even prevent the discharge of the mucus.This leads to a blockage in the sinuses associated with viscous mucus,which leads to a deterioration of function or loss of function of thecilia. This ultimately causes a deterioration of the cleaning mechanismof the mucous membrane.

Such a microenvironment promotes the rapid increase of the ubiquitousmicroorganisms. Over a relatively long period of time, theseunfavourable conditions, such as a swollen mucous membrane and ciliaconglutinated by viscous mucus, this may lead to chronic sinusitis,resulting in permanent damage to the mucous membrane and to the ciliatedepithelium. Pathogens relevant to the airways, which are also to beunderstood in particular to include ENO-relevant germs, which settle inthe mucus, for example include Staphylococcus aureus, Staphylococcusepidermidis, Streptococcus pyogenes, Streptococcus pneumoniae,Streptococcus mutans or Haemophilus influenzae.

In the event that the upper airways become conglutinated as a result ofviscous mucus, the ingredients of the used composition (Gentianalutea:Rumicis herba:Verbena officinalis:Sambucus nigra:Primulaveris=1:3:3:3:3) induce the formation of fresh, thin mucus, whereby theabove-described process of expectoration and the transporting away andalso reduction of the inflammatory symptoms is achieved and a healingprocess of the nasal mucous membrane is initiated. Sinupret® gentlycauses the re-establishment of the self-cleaning power of the airwaysand simultaneously develops a strong antimicrobial effect. Sinupret® ischaracterized by its good compatibility, composition established byBIONORICA and dosing, which rarely cause side effects in patients, and,in addition, no interactions with other pharmaceuticals are known.

Dry plant extracts are described in general and dry plant extractsformed from aqueous/ethanolic extracts are known.

Dry plant extracts can be produced for example in large amounts inaccordance with the technical teaching of EP0753306.

However, there is a considerable need to provide a new Sinupret® dryplant extract, which has an advantageous improved effect of the plantcombination.

The Deutsche Arzneimittelbuch (DAB or German Pharmacopoeia) 2010establishes minimum contents of ingredients for drug quality, such thatincreased efforts have to be made for constant and improved quality.Extraction and drying methods specifically constitute a bottleneck for asufficient quality of phytopharmaceuticals.

BRIEF SUMMARY OF THE INVENTION

Based on this prior art, the object of the present invention istherefore to provide an improved method for producing a dry plantextract and also an improved dry plant extract as such, which containsat least one ethanolic/aqueous extraction step, wherein the plants areselected from the group consisting of: Rumex acetosa L., Rumexacetosella L, Rumex obtusifolius L., Rumex patientia L., and Rumexcrispus L., (referred to hereinafter and in the claims by the collectiveterm “Rumicis herba”); Verbena officinalis; Sambucus nigra; Primulaveris; and/or Gentiana lutea and mixtures thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibition of human flu virus and pig flu virusproliferation.

FIG. 2 shows the inhibition of respiratory virus proliferation.

FIG. 3 shows the effect in the inflammation model.

FIGS. 4A-4D show a comparative illustration by means of graphs.

FIG. 5 shows the effect of Sinupret® drug mixture (SIN) and of Sinupretdry extract (SIN TR) on the expression of COX-2 protein in the rat lung.

FIG. 6 shows the effect of Sinupret® drug mixture (SIN) and Sinupret dryextract (SIN TR) on cytokines.

FIG. 7A shows that the dry extract according to the inventiondemonstrates a change of the transepithelial short-circuit current (ISC)after addition of the amiloride and forskolin.

FIG. 7B shows that a dose-dependent rise in the cilial beat frequency(CBF) measurements can be observed, which is accompanied by a chlorideion secretion.

FIG. 8 shows paw volumes determined in all animals by means ofplethysmography before (−1 h) and after carrageen injection (+1 h).

FIG. 9 shows paw volumes determined in all animals by means ofplethysmography before (−1 h) and after carrageen injection (+15 min.,+30 min., and +1 h).

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, an improved dry plant extract could be obtained by meansof a twofold extraction process, wherein, in a first step, anaqueous/ethanolic extraction took place, and, in a second step, anaqueous extraction took place.

Surprisingly, the content of advantageous active ingredients in existingactive ingredient mixture in the respective overall extract can beenriched after drying by means of this method according to theinvention, such that an improved pharmacological efficacy of theobtained dry plant extract is achieved.

The invention therefore relates to a method for producing dry plantextracts, said method comprising the following steps:

-   -   a.) alcoholic/aqueous extraction of Rumicis herba, Verbena        officinalis, Sambucus nigra, Primula veris and Gentiana lutea,    -   b.) separation of the supernatant,    -   c.) second aqueous extraction of the residue from a.),    -   d.) separation of the supernatant,    -   e.) combining of the obtained supernatants from b.) and d.),    -   f.) drying of the supernatants and provision of the dry plant        extract.

The improvement according to the method lies in the fact that a quickerefficacy is achieved compared to a conventional simple aqueous-ethanolicextract (see the examples). This is surprising since a linearextrapolation would have been expected at best as a result of a changeof the solvent for extraction (for example if aqueous/ethanolicextraction is carried out exclusively or if aqueous extraction iscarried out exclusively).

In particular, the method according to the invention allows an improveddosing, such that an increased curative effect can be achieved with thesame dose.

The invention therefore likewise relates to a dry plant extract, whichis obtained or is obtainable in accordance with a method according tothe invention (hereinafter dry (plant) extract according to theinvention, Sinupret dry extract (TE)).

In a further preferred embodiment of the method according to theinvention, the ratio of Gentiana lutea:Rumicis herba:Verbenaofficinalis:Sambucus nigra:Primula veris is 1:3:3:3:3, in each case+/−0.3 to 0.5 (for example 1:3.2:2.9:2.5:3.2:3).

Furthermore, it is preferable for a plant grouping of all plant (drugs)to be provided in a batch, wherein the ratio of plant (drugs) is asmentioned above. Furthermore, it is preferable for the presented plant(drugs) to be cleaned and cut.

In a further preferred embodiment of the method according to theinvention, the aqueous/alcoholic extraction agent in step a.) has acontent 40:60 (v/v) to 60:40 (v/v), in particular 41:59 (v/v) or 50:50(v/v) of water/alcohol. Ethanol is preferred, however methanol andpropanol or mixtures thereof are also included. Furthermore, the use of96% ethanol is preferred.

In accordance with the invention, the extraction in steps a.) and c.) isalso carried out at 20 to 40° C., wherein the extraction is carried outin steps a.) and c.) in 2 to 8 h.

Within the meaning of this invention, a “separation of the supernatant”can be carried out continuously or discontinuously after extraction inaccordance with the invention by means of draining, decantation,filtration, screening or a separation method known to a person skilledin the art.

Furthermore, it is preferable for the drying according to step f.) to becarried out under vacuum at 30 to 60° C., in particular at 40 to 50° C.,preferably in a vacuum agitated dryer. The dry extract according to theinvention has a residual ethanol content of at most 0.5%.

Further suitable drying methods according to the invention will beexplained:

Dry plant extracts are produced conventionally, wherein a plant materialis extracted with the aid of a solvent or solvent mixture, for exampleby means of maceration or percolation, and, after separation of theextraction residue, the obtained fluid extract or the obtained tinctureis compressed until dry.

Conventional drying methods are comprised in accordance with theinvention and include fluidized bed drying or the compression to a thickor spissum extract and subsequent vacuum band drying or tray drying ofsaid spissum extract.

Conventional methods for producing dry plant extracts according to theinvention via a fluid extract (or liquid plant extract) or a tincturemay likewise be considered; wherein, after subsequent distillation ofthe solvents, what is known as a spissum extract (viscose extract) isobtained, to which auxiliary agents and/or additives, such as lactose,polyvinylpyrrolidone, sucrose, silicon dioxide, etc., are often added.This moist, viscose mass is then introduced into tray cabinets or dryersfor desired dry extract preparation.

A method that is very often used in dry extract production is what isknown as the vacuum band drying method. In this case, the spissumextract is brought to dry extract preparation after preliminary dryingvia a downdraft vaporizer.

A drying method by means of a fluidized bed dryer requires temperaturesbetween approximately 47° C. and 117° C. The drying process in thismethod is carried out under normal pressure conditions.

A gentle drying method for obtaining dry plant extracts according to theinvention is described in EP 0 753 306. In accordance with the describedmethod, the fluid extract from the plant materials obtained in theextraction is introduced in accordance with the method according to theinvention in a vacuum drying system, preferably a vacuum agitated dryerwith a multi-branched stirrer extending through a cylindrical mixing anddrying chamber and having its own drive, and, where necessary, providedwith vapour filter, backwash device, solvent condenser with aftercoolerand collection vessel, back-condenser and a process, control andregulation unit, and optionally with granulation nozzles, and is driedin the dryer equipped with a chopper extending over the overall depth ofthe mixing and drying chamber and having blades rotating through acomb-shaped stator at a supply and return temperature between 120° C.and 5° C., an inner chamber temperature between 10° C. and 80° C., afilter temperature from 15° C. to 55° C., and a pressure between 0.51,000 mbar, and also a stirrer speed of rotation from 0 and 10 rpm and achopper speed of rotation between 200 and 800 rpm. The vacuum dryingsystems used according to EP 0 753 306 are sold for example by theformer companies Firmen Inox Glatt AG or Inox-Maurer AG under the names“IUT” or “INOX”. Current manufacturers and distributors include, forexample, De Dietrich Process Systems GmbH, Mainz, Germany (Rosemund®).

With this vacuum drying system, the fluid extract to be dried is pumpedfrom above into the mixing and drying chamber in the batch method and isthen subjected to a vacuum.

A preferred vacuum drying system comprises the following features, asare implemented for example in a known IUT/INOX system (see above):

a.) A multi-branched stirrer extending through a cylindrical mixing anddrying chamber and having its own drive and, depending on requirements,vapour filter, backwash device, solvent condenser with aftercooler andcollection vessel, back-condenser, a process, control and regulationunit, and optionally granulation nozzles.b.) Furthermore, a chopper extending over the entire depth of the dryingand mixing chamber and having a drive independent of the stirrer may beprovided as well as optionally a comb-like stator for increasing thechopper effect.c.) Furthermore, one or more nozzles may optionally be provided in orderto introduce the liquid plant extract from a reservoir into the dryingchamber, for example as described in WO2002073108.

The dry plant extracts obtained in this way are processed further toform pharmaceutical preparations.

The agent having an antimicrobial effect, containing the dry plantextract according to the invention, can thus advantageously be used inthe treatment of infections triggered by pathogens relevant to theairways. The expectorant and anti-inflammatory effect is supplemented bythe additional antimicrobial effect. An infection of the upper airwaysis thus confined or even completely stopped in addition to the looseningof the viscous mucus charged with pathogens, weakened by killing and/orreduction of the proliferation of the bacterial pathogens.

Due to the above-described invention, a patient suffering from sinusitisand/or rhinosinusitis and/or inflammation of the nasal sinuses forexample, in particular in the acute form in each case, is treated in agentle manner without the use of synthetic-chemical components.

The (pharmaceutical) composition of the present invention having anantimicrobial effect is particularly effective against pathogensrelevant to the airways, wherein it has demonstrated antimicrobialefficacy specifically against gram-positive cocci, such asStaphylococcus aureus, Staphylococcus aureus (MRSA), Staphylococcusepidermidis, Streptococcus pyogenes, Streptococcus pneumoniae andStreptococcus mutans, against gram-negative bacilli, such as Haemophilusinfluenzae, and against Enterobacteriaceae faecalis. Efficacy againstviruses is also achieved.

The galenic formulation of the antimicrobial agent can be selected fromthe group consisting of: drops, juice, syrup, tablets, dragées,capsules, retard formulations, rectal or vaginal suppositories,infusions, in particular throat sprays and disinfection solutions;ointments, emulsions, granulates, powders, nose sprays, liquid or solidpreparations for inhalation, compresses, waddings, in particular woundand gum dressings, tamponades, including for teeth, rinsing solutions,in particular in combination with physiological and hyperosmolarconcentrations of salts or salt mixtures, preferably table salt, inparticular sea salt. Of course, the formulation can containpharmaceutically conventional auxiliary agents.

The present invention therefore also relates to the use or applicationof the antimicrobial agent according to the invention containing a dryplant extract according to the invention for treatment of infectionstriggered by pathogens relevant to the airways, and to the use forproduction of a pharmaceutical, and also to a pharmaceutical as such.

In a further embodiment, the invention relates to a pharmaceutical foruse or application in the case of sinusitis and/or rhinosinusitis and/orinflammation of the nasal cavities, in particular in the acute form ineach case, in particular for treatment and prophylaxis of sinusitisand/or rhinosinusitis and/or inflammation of the nasal sinuses, inparticular in the acute form in each case.

Due to the ENO relevance of the tested pathogens, the agent according tothe invention is likewise outstandingly suitable in any instance inwhich bacteria can be fought directly and immediately locally ortopically.

The agent according to the invention can thus preferably be used (inaddition to the systemic application) directly at the contaminatedlocation, for example in the form of a disinfection solution to bebiologically degraded 100%, or of course also for direct topicalapplication to skin and mucous membranes both in humans and in animals.Different forms of the application are considered for this purpose. Theformulations are particularly suitable as solutions, creams, ointmentsand emulsions in the dermatological field of human and also veterinarymedicine. Here, the agent according to the invention can be applieddirectly to the diseased part of the skin and/or can be used in the formof saturated compresses, waddings or tamponades.

Of course, the application of the agent according to the invention inthe overall field of diseases of the entire respiratory tract, inparticular of the upper airways, here preferably in the region ofthroat, nasal and nasal sinus mucous membranes, is of particularinterest. Nasal rinsing, in particular in conjunction with salts, forexample in combination with a physiological or hyperosmolar saltsolution, is of particular significance. In accordance with theinvention, a nasal spray containing the agent according to the inventionis also included.

The broad range of new application possibilities reaches from tonsilpaint solutions and gargling solutions in the case of pharyngealinfections to powder inhalation preparations or nebulizer inhalationpreparations.

Further fields of application and indication comprise wound and gumdressings, for example in the form of cotton waddings or cotton yarnwaddings, which are saturated with the agent according to the invention.Ear rinses with solutions that contain the agent according to theinvention are also conceivable in the case of infections of the earcanal.

The invention therefore likewise relates to a pharmaceutical for use andapplication of diseases of the entire respiratory tract, in particularof the upper airways, in particular in the region of the throat, nasaland nasal sinus mucous membranes, respiratory diseases, in particularmucoviscidosis (cystic fibrosis), in particular the treatment andprophylaxis thereof. Mucoviscidosis can particularly advantageously betreated, see FIGS. 7A and 7B.

A further preferred embodiment concerns a dietary supplement containingthe agent according to the invention, in particular in the form of adietary composition. Suitable foods or foodstuffs, including water,according to the invention are those as defined (although notdefinitively) for example in regulation (EC) No. 178/2002 of 28 Jan.2002, such as bakery products and beverages and infant foodpreparations. The dietary supplement according to the invention can bemixed with a suitable physiologically compatible carrier.

The pharmaceutical preparations according to the invention can beproduced in the form of dosage units. This means that the preparationsmay be present in the form of individual parts, for example capsules andampoules, of which the active ingredient content of dry plant extractscorresponds to a fraction or a multiple of an individual dose. Thedosage units may contain, for example, 1, 2, 3 or 4 single doses or ½, ⅓or ¼ of a single dose. A single dose preferably contains the amount ofdry plant extract (active ingredient) according to the invention that isadministered in one application and that usually corresponds to theentire daily dose or one half, one third or one fourth of a daily dose.A dosage of three times daily, preferably in the form of a tablet, inparticular in the morning, afternoon and evening, possibly at mealtimes, is preferred.

In a further preferred embodiment, the galenic formulation of a calciumcoated tablet can be selected, as disclosed in EP1392337.

Non-toxic, inert pharmaceutically suitable carrier substances are to beunderstood to include solid, semi-solid or liquid diluting agents,fillers and formulation aids of any type, such as a) fillers anddiluting agents, for example starches, lactose, cane sugar, glucose,mannite, dextrins, maltodextrin and silicic acid, highly dispersedsilicon dioxide, b) binders, for example carboxymethyl cellulose,cellulose powder, microcrystalline cellulose, alginates, gelatines,polyvinylpyrrolidone, c) humectants, for example glycerol, d) splittingagents, for example agar-agar, calcium carbonate and sodium carbonate,e) dilution restrainers, for example paraffin, and f) resorptionaccelerators, for example quaternary ammonium compounds, g) wettingagents, for example cetyl alcohol, glycerol monostearate, h) adsorptionagents, for example kaolin and bentonite, and i) lubricants, for exampletalc, calcium stearate and magnesium stearate, and solid polyethyleneglycols or mixtures of the substances listed under a) to i).

The tablets, dragées, capsules, pills and granulates may be providedwith the conventional coatings and coverings, optionally containingopacity promoting agents, for example such as, although notdefinitively, hypromellose, microcrystalline cellulose, stearic acid,titanium dioxide, and may also be composed such that they release theactive ingredient(s) only, or preferably, in a certain part of theintestinal tract, in a delayed manner where necessary, wherein polymersubstances and waxes for example may be used as embedding matter.

EXAMPLES

These examples are intended merely to explain the invention, withoutlimiting the invention to these examples.

Hereinafter, “dry extract (TE)” means the dry plant extract producedaccording to the invention.

Example 1

The antiviral activity of the dry extract according to the invention wasconfirmed in a number of in vitro tests.

In these tests, the general cell-damaging (cytotoxic) effect of the dryextract according to the invention compared to the known productSinupret® (alcoholic/aqueous) was initially tested. After incubation ofsuitable cell lines (for example HeLa, HEp-2) with different virusesover a period of one hour, the infected cell lines were treated withdifferent concentrations, and the effect on the virus proliferation wasthen measured.

The antiviral activity was determined quantitatively in vitro via thedetection of a cytopathogenic effect (Adeno5 Virus), in a plaquereduction assay (FluA, HRV14, RSV) and in virus-specific enzymeimmunoassays (ELISA; Adeno5, RSV).

In the event of detection of a cytopathogenic effect, thevirus-sensitive cells grown confluently were infected with a definedvirus solution (M.O.I., multiplicity of infection). After incubation forone hour, the virus inoculum was drawn off and the infected cell layerswere washed. The physiological substance concentrations were then added.The respective test batches were cultivated until a 70-90%cytopathogenic effect (CPE), which presents itself as a destroyed cellregion, was observed under microscope in the untreated virus controls.The area of the destroyed cell region was defined as 100% infection.Comparatively, the cell areas of the respective test batches wereevaluated so that inhibiting effects of the substances to be analysedcan be shown as an inhibition percentage (% inhibition).

In the case of the plaque reduction assay, the virus-sensitive cellsgrown confluently were infected with a defined virus solution (M.O.I.,multiplicity of infection). After incubation for one hour, the virusinoculum was drawn off and the infected cell layers were washed. Thephysiological substance concentrations and also a solid medium component(agarose or methylcellulose) were then added, followed by furtherincubation. The infection area was delimited by the solid component inthe superimposed medium, such that a series of infected cells (“plaque”)was produced. The respective test batches were cultivated until the setplaque number (M.O.I.) was observed under microscope in the untreatedvirus controls. By fixing and dyeing the cell layer, the virus plaquecan be made visible as light rings in the dark-coloured cell layers. Theplaque number was determined with the aid of image processing systems.The plaque number of the untreated control was defined as 100%infection. By contrast, the plaque number of the respective test batcheswas evaluated such that inhibitory effects of the test substance can bepresented as an inhibition percentage (% inhibition).

The virus production was analysed by ELISA. Test strips with antibodiesagainst the specific viruses bind the viruses found in the cell culturesupernatant of the infected cell lines. In order to make the reactionvisible, a pathogen-specific detection antibody labelled with peroxidasewas introduced. After addition of a substrate/chromogen and also ofhydrogen peroxide and tetramethylbenzidine, a colour reaction takesplace. The intensity of the colouration was determined photometricallyand was proportional to the content of virus antigen. The virusproduction in the case of infected and treated cells was analysed afterinfection of virus-sensitive cells grown confluently with a definedvirus solution (M.O.I., multiplicity of infection). After incubation forone hour, the virus inoculum was drawn off and the infected cell layerswere washed. The physiological substance concentrations were then added.The respective test batches were cultivated until a 70-90%cytopathogenic effect (CPE) was observed under microscope in theuntreated virus controls. The newly synthesized viruses were located inthis stage in the cell culture supernatant. The photometricallydetermined extinction values of the untreated controls were defined as100% infection. By way of comparison, the extinction values of therespective test batches were evaluated, such that inhibitory effects ofthe test substances can be presented as an inhibition percentage (%inhibition).

A significant inhibition of the virus proliferation, that is to say areduction of the virus load, was demonstrated in all tests (see FIG. 1).

The dry extract according to the invention inhibits the proliferation ofhuman (FluA) and pig (pFluA) influenza viruses (flu viruses).Concentrations of 124.8 μg of Sinupret/ml (FluA) and of 43.4 μg ofSinupret/ml (pFluA) are sufficient in order to inhibit (reduce) thevirus (load) by 50%. The Sinupret dry extract according to the inventiondemonstrated a lower EC50 against virus strains HRV 14, Adeno5 and RSV,and therefore accordingly a greater efficacy compared to Sinupret®alcoholic/aqueous (see FIGS. 1 and 2).

TABLE 0 antiviral effect Sinupret Sinupret alcoholic/aqueous dry extractVirus strain EC50 [μg/ml] EC50 [μg/ml] HRV 14 73.1 50.5 Adeno 5 66.413.8 RSV 20.7 10.4

Example 2

The anti-inflammatory activity of Sinupret could be confirmed in theanimal model. For example, carrageen-induced paw oedema in rats (MaleWistar Han rats, 220-230 g) was selected as the test model. In thismodel, it was possible to examine the anti-inflammatory effect of testsubstances by measuring their inhibitory effect on the paw oedema orpleuritis caused by carrageen. The following were used as referencesubstances: (RS)-2-[4-(2-methylpropyl)phenyl]propanoic acid (Ibuprofen®)and 2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid(indomethacin, “indo”). In the present tests, groups of 10 rats in eachcase were treated either with Ibuprofen®, indomethacin, Sinupret® dryextract (SIN TR) or Sinupret® drug mixture (SIN, as commerciallyavailable) and were injected one hour later with carrageen. Theinhibition of the oedema formation by the test and reference substanceswas determined at various moments in time after carrageen injection,wherein the paw volume of animals treated only with carrageen was usedas a control (vehicle=blank control). The results of these tests arepresented in Tables 1 and 2 and in FIGS. 3 to 6 and will be explainedhereinafter.

FIG. 3: In the carrageen model, it was found that the dry extractaccording to the invention inhibits the paw oedema induced by carrageenafter just 30 minutes following carrageen injection, and morespecifically inhibits said paw oedema to a greater extent than theSinupret drug mixture, where as Ibuprofen® still did not show anyanti-inflammatory effect at this early moment in time. Even one and twohours after oedema induction, the anti-inflammatory effect of the dryextract according to the invention was still stronger than that ofIbuprofen® and Sinupret® drug mixture (SIN).

Table 1 shows the effect of Sinupret® (SIN) in the case ofcarrageen-induced pleuritis on the basis of the inflammatory markers(PGE₂, LTB₄, TNF alpha, IL1 beta) with measurement after 4 h.

Rats (10 per group) were each treated with 100 mg/kg or 500 mg/kg of SINand by way of comparison with 5 mg/kg of indomethacin and blank and wereinjected 1 hour later with carrageen.

“Inflammatory cells” correlates to PMN (polymorphonuclear neutrophils)accumulation/infiltration.

Statistics: average+/−SEM, n=10, **p<0.01; ***p<0.001 vs vehicle (blank)(Tukey Test), p<0.05 is statistically significant.

A comparative illustration by means of graphs is provided in FIG. 4.

exudate Inflammatory PGE₂ LTB₄ TNFα IL1β treatment volume (ml) cells ×10⁶ ng/rat ng/rat ng/rat ng/rat vehicle 0.196 ± 0.012 38.6 ± 2.54 0.861± 0.061 0.50 ± 0.07 4.94 ± 0.33 3.37 ± 0.25 Sinupret ® 0.125 ± 0.01827.7 ± 2.61 0.973 ± 0.066 0.61 ± 0.06 5.94 ± 0.30 2.45 ± 0.23 100 mg/kg** ** (27%) (36%) (28%) Sinupret ® 0.034 ± 0.010 23.5 ± 1.34 0.865 ±0.065 0.67 ± 0.06 5.48 ± 0.19 2.03 ± 0.30 500 mg/kg *** *** ** (83%)(39%) (40%) Indometharin 0.009 ± 0.003 11.4 ± 0.93 <0.125 0.43 ± 0.025.48 ± 0.28 3.17 ± 0.18 6 mg/kg *** *** *** (96%) (70%)

Table 2 shows the effect of Sinupret® dry extract (SIN TR) in the caseof carrageen-induced pleuritis on the basis of the inflammatory markers(PGE₂, LTB₄, TNF alpha, IL1 beta) with measurement after 4 h.

Rats (10 per group) were each treated with 100 mg/kg or 500 mg/kg of SINTR and by way of comparison with 5 mg/kg of indomethacin and blank andwere injected one hour later with carrageen.

“Inflammatory cells” correlated with PMN (polymorphonuclear neutrophils)accumulation/infiltration.

Statistics: average+/−SEM, n=10, **p<0.01; ***p<0.001 vs vehicle (blank)(Tukey Test), p<0.05 is statistically significant.

exudate Inflammatory volume cells PGE₂ LTB₄ TNFα IL1β treatment ml ×10⁶ng/rat ng/rat ng/rat ng/rat vehicle 0.196 ± 0.012 38.6 ± 2.54 0.861 ±0.061 0.50 ± 0.07 4.94 ± 0.33 3.37 ± 0.21 dry extract 0.112 ± 0.009 22.8± 2.19 0.775 ± 0.050 0.41 ± 0.05 5.36 ± 0.44 3.00 ± 0.21 100 mg/kg ****** (10%) (11%) (43%) (41%) dry extract 0.072 ± 0.013 18.8 ± 0.92 0.603± 0.039 0.64 ± 0.05 4.66 ± 0.30 2.74 ± 0.09 500 mg/kg *** *** ** (19%)(63%) (51%) (30%) Indometharin 0.009 ± 0.003 11.4 ± 0.93 <0.125 0.43 ±0.02 5.48 ± 0.28 3.17 ± 0.18 6 mg/kg *** *** *** (96%) (70%)

A comparative illustration by means of graphs is provided in FIG. 4.

FIG. 5 shows the effect of Sinupret® drug mixture (SIN) and of Sinupretdry extract (SIN TR) on the expression of COX-2 protein in the rat lung.

Rats (10 per group) were each treated with 100 mg/kg or 500 mg/kg of SINor SIN TR and by way of comparison with 5 mg/kg of indomethacin andblank and were injected 1 hour later with carrageen. A western blot wascarried out in each case with 30 μg of protein from rat lung(homogenized) on 10% SDS polyacrylamide gel and COX-2 was analysed.

FIG. 6 shows the effect of Sinupret® drug mixture (SIN) and Sinupret dryextract (SIN TR) on cytokines.

Rats (10 per group) were each treated with 100 mg/kg or 500 mg/kg of SINor SIN TR and by way of comparison with 5 mg/kg of indomethacin andblank and were injected 1 hour later with carrageen. The inflammatorymarkers IL1 beta and TNF alpha were determined 4 h after carrageeninjection.

Statistics: average+/−SEM, n=10, **p<0.01; ***p<0.001 vs vehicle (blank)(Tukey Test), p<0.05 is statistically significant.

Conclusion:

The Sinupret dry extract (SIN TR) according to the inventiondemonstrated, at least for PGE2, a particularly advantageous significantinhibition of PGE2 formation (30%; p<0.01; FIG. 4C, Table 2) compared toSinupret® drug mixture (SIN). Furthermore, the Sinupret dry extract (SINTR) according to the invention is more effective at lower dosagecompared to the known Sinupret® drug mixture (SIN).

Example 3

The following example shows that the dry extract according to theinvention, with topical use, activates chloride secretion, most likelyvia the activation of CFTR. In addition, the dry extract according tothe invention stimulates the ciliary beat frequency.

Material: cell culture: human bronchial epithelial cells (HBE) wereacquired from Lonza (Walkersville, Md.) and were expanded with bronchialepithelial cell growth medium (BEGM) from Lonza (Walkersville, Md.). 250mg of dry extract according to the invention were dissolved in 1 ml of50% ethanol and were treated by ultrasound at 35 kHz for 30 minutes withsubsequent centrifugation at 3,000 g for 10 minutes at room temperature.The supernatant was suctioned off. Amiloride (Sigma, St. Louis, Mo.) wasdissolved in distilled deionized water and diluted 1,000 times.Forskolin (Kaliokemm, EMD, San Diego, Calif.) was dissolved in DMSO anddiluted 1,000 times.

An Ussing chamber (Physiology Instruments San Diego, Calif., USA) wasused, containing Transwell inserts (Corning Life Sciences) withimplementation at 37 degrees and a monolayer with a voltage terminal of0 volts (VCC 600) (Physiology Cal Instruments San Diego, Calif., USA)after setting of the forward resistance. Transwell filters were placedin the solution at 37 degrees and the solution was infused continuouslywith 95% oxygen to 5% CO₂. The transepithelial resistance (RT) wasadjusted using a computer program (Physiology Instruments San Diego,Calif., USA) at 640 ms, bipolar 10 mV potential via the monolayermeasured in accordance with Ohms Law. By definition, a positive resultis an anion secretion or cation absorption. The experiments wererepeated at least 3 times in HBE cell cultures.

Used electrolyte solution (in mM): 120 NaCl, 25 NaHCO₃, 3.3 KH₂PO₄, 0.8K₂HPO₄, 1.2 MgCl₂, 1.2 CaCl₂ and 10 glucose.

The cilial beat frequency (CBF) measurements were carried out inaccordance with a method according to Woodworth et al (Woodworth, B A,Zhang S, Tamashiro E, Zinc increases ciliary beat frequency in a calciumdependent manner, Am J Rhinol Allergy 24: 6-10, 2010). The images werecreated by Leica Microsystems, Inc., Bannockburn, Ill. with a 63× lens(model A 602f-2, high-speed monogram digital video camera, Basler AG,Ahrensburg, Germany). The dry extract according to the invention wasexamined for transepithelial electrolyte transport. The dry extract wasapplied in increasing concentration amounts to the basolateral surfaceof HBE cells in the Ussing chamber, before subsequent addition ofamiloride and forskolin.

In FIGS. 7a and 7b , the dry extract according to the inventiondemonstrates a change of the transepithelial short-circuit current(I_(SC)) (7A) after addition of amiloride and forskolin, such that adose-dependent rise in the cilial beat frequency (CBF) measurements canbe observed (7B), which is accompanied by a chloride ion secretion.

These results prove the advantageous use of the dry extract according tothe invention, for example by means of a nasal spray, since improvedmucociliary clearance (MCC) can be achieved.

Conclusion: The dry extract according to the invention is suitable forthe treatment of respiratory diseases, in particular for the treatmentof mucoviscidosis (cystic fibrosis).

Example 4

The anti-inflammatory effect of the Sinupret dry extract according tothe invention and Sinupret drops (alcoholic/aqueous (“Sinupret OD”)) wasexamined as follows.

Carrageen-induced paw oedema (see above): The test animals (n=8/group)were weighed in the fasted state and the basal volume of the rear pawwas measured. The animals were fed the test substances (10 mL/kg of bodymass) (Sinupret dry extract (TE): 5 mg/kg, equivalent to the amount ofdrug mixture in 1 mL of drops/kg; 50 mg TE/kg corresponding to 1 timesthe human equivalent dose (1×HED); Sinupret drops (Sinupret OD): 1 mLOD/kg, corresponding to 1 times the human equivalent dose (1×HED); 2.5mL OD/kg, equivalent to the amount of drug mixture in 50 mg TE/kg;indomethacin: 20 mg/kg as a positive control; 10% v/v ethanol as vehiclecontrol). After 60 minutes, the animals were provoked by a subcutaneousinjection of carrageen (0.1 mL of a 1% w/v solution in physiologicaltable salt solution) into the left rear paw (in a plantar manner). Pawvolumes were determined in all animals by means of plethysmographybefore (−1 h) and after carrageen injection (study 1: +1 h (FIG. 8),study 2: +15 min, +30 min, +1 h (FIG. 9)). The inhibition percentage ofpaw swelling vs. vehicle control was calculated individually for eachtest animal.

The results are shown by way of comparison in FIG. 8 (inhibition ofinflammation after 1 hour).

FIG. 8: A: Sinupret TE and Sinupret OD were each administered in a doseequivalent to 1 times the human equivalent dose. Sinupret TE has aquicker and stronger anti-inflammatory effect than Sinupret OD. Theinhibition of inflammation by Sinupret TE, not by Sinupret OD, iscomparable after 1 h with the inhibitory effect of the knownantiphlogistic indomethacin.

B, C: The dose administered here of Sinupret TE and Sinupret OD iscomparable based on the respective amount of drug mixture (DM) used forproduction (B: 23.6 mg/kg, C: 223 mg/kg). Sinupret TE has a quicker andstronger anti-inflammatory effect compared to Sinupret OD. Theinhibition of inflammation produced by Sinupret TE, not by Sinupret OD,is comparable after 1 h with the inhibitory effect produced by the knownantiphlogistic indomethacin.

The results are shown by way of comparison in FIG. 9 (inhibition ofinflammation after +15 min, +30 min, +1 h).

FIG. 9: the administered dose of Sinupret TE and Sinupret OD iscomparable based on the respective amount of drug mixture used forproduction (23.6 mg of drug mixture/kg). Sinupret TE is characterized byan earlier and stronger onset of action compared to Sinupret OD.Sinupret OD only inhibits inflammation from 30 min. Sinupret TE inhibitsinflammation comparatively strongly and quickly over the entire periodof time tested in a manner comparable to the known antiphlogisticindomethacin.

Example 5

The following example describes specific marker compounds from themulti-substance mixture.

Implementation:

All samples were extracted immediately after the individual extractionsteps, were filtered and were analysed by mass spectrometry in aconcentration of 600 mg/L (in 30 vol. % MeOH). Methylparaben was used asan internal standard. The samples were processed twice and analysedtwice. The following parameters and devices were used:

MS: 5600 Triple ToF (ABSciex); HPLC: Agilent 1290

Software: Analyst TF 1.5.1, MultiQuant 2.1.1, MarkerView 1.2.1

Stationary phase: Zorbax RRHD Eclipse Plus C18, 2.1×50 mm, 1.8 μm

LC Method:

Time Flow rate Step (min) (μL/min) Eluent A Eluent B 0 0.00 600 95.0 5.01 1.00 600 95.0 5.0 2 6.00 600 69.0 31.0 3 10.00 600 0.0 100.0 4 11.00600 0.0 100.0 5 11.10 600 95.0 5.0 6 13.00 600 95.0 5.0 A = 0.1% offormic acid in H2O; B = acetonitrileMS Method:

Scan Type negative TOF MS Duration 10.997 mins Cycle Time 0.2750 secsGS1 (Spray Gas): 70.00 GS2 (Turbo Gas): 55.00 CUR (Curtain Gas): 25.00TEM (GS 2): 500.0 ISVF (Ion Spray Voltage 4500.0 Floating): CAD(Collision Gas): 6 TOF Masses (Da): 130-2000 Accumulation Time (sec):0.25 Time Bins to sum: 4 DP: −100.0 CE: −10.0

TABLE 3 selective enrichment and depletion of characteristic ingredientsm/z = 399.2 m/z = 540.3 m/z = 279.2 RT = 0.5 min RT = 8.4 min RT = 9.6min Lab batch 1^(st) extraction (59 vol. % EtOH)  8% 2879%  737.2 g(±11.7 g) 2^(nd) extraction (water) 224%  61%  0.0 g total extract 100%100% 622.3 g (±6.0 g) Production 1^(st) extraction (59 vol. % EtOH)16.5%  896% 745.5 g (±19.6 g) batch 2^(nd) extraction (water)  67% 104% 0.0 g total extract 100% 100% 119.9 g (±12.2 g)

In so far as quantified via reference standards, the quantities ofingredients are specified as absolute content in [g]. Otherwise, thecontent of ingredients is specified in relation [%] to the respectivetotal extract (after step f.)). The data originate from a lab batch A1and a production batch P1. The signals were detected with negativeionisation.

TABLE 4 ingredients with typical enrichment in the first extraction stepwith increase by subsequent aqueous extraction. m/z = 401.1 m/z = 463.1m/z = 623.2 RT = 2.6 min RT = 3.7 min RT = 3.9 min Lab batch 1^(st)extraction (59 vol. % EtOH) 500.7 g (±6.4 g) 122.0 g (±2.3 g) 575.6 g(±6.4 g) 2^(nd) extraction (water) 152.3 g (±3.6 g) 11.9 g (±0.4 g) 91.2g (±0.1 g) total extract 655.4 (±3.3 g) 134.2 g (±0.8 g) 657.6 g (±17.4g) Production 1^(st) extraction (59 vol. % EtOH 704.5 g (±6.1 g) 127.6(±3.0 g) 527.0 g (±12.5 g) batch 2^(nd) extraction (water) 70.0 g (±0.03g) 9.0 g (±0.02 g) 42.3 g (±1.7 g) total extract 788.2 g (±11.8 g) 138.1g (±0.7 g) 571.1 g (±20.2 g)

In so far as quantified by reference standards, the quantities ofingredients are specified as absolute content in [g]. Otherwise, thecontent of ingredients is specified in relation [%] to the respectivetotal extract (after step f.)). The data originate from the lab batch A1and from the production batch P1. The signals were detected withnegative ionization.

The invention claimed is:
 1. A method for producing a dry plant extractmixture, said method comprising the following steps: a) a firstextraction of Rumicis herba, Verbena officinalis, Sambucus nigra,Primula veris and Gentiana lutea with water/alcohol to produce a firstsupernatant; b) separating of the first supernatant to form a residue;c) a second aqueous extraction of the residue to form a secondsupernatant; d) separation of the second supernatant; e) combining thefirst and second supernatant; and f) drying of the first and secondsupernatant to form the dry plant mixture.
 2. The method of claim 1,wherein the ratio in step a.) of Gentiana lutea:Rumicis herba:Verbenaofficinalis:Sambucus nigra:Primula veris is 1:3:3:3:3.
 3. The method ofclaim 1, wherein, in step a.), the water/alcohol has a content of 40:60(v/v) to 60:40 (v/v) water/alcohol.
 4. The method of claim 1, wherein,in step a.), the water/alcohol has a content of 41:59 (v/v)water/alcohol.
 5. The method of claim 1, wherein, in step a.), thewater/alcohol has a content of 50:50 (v/v) water/alcohol.
 6. The methodof claim 1, wherein the water/alcohol comprises ethanol.
 7. The methodof claim 1, wherein the water/alcohol comprises 96% ethanol.
 8. Themethod of claim 1, wherein the plants in a.) are provided together in abatch.
 9. The method of claim 1, wherein the extraction in steps a.) andc.) is carried out at 20 to 40° C.
 10. The method of claim 1, whereinthe extraction in steps a.) and c.) is carried out in 2 to 8 h.
 11. Themethod of claim 1, wherein the drying process of step f.) is carried outunder vacuum at 30 to 60° C.
 12. The method of claim 1, wherein thedrying process of step f.) is carried out under vacuum at 40 to 50° C.13. The method of claim 1, wherein the drying process of step f.) iscarried out in a vacuum agitated dryer.